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A R&S 002397 UnscKcfuled induced by f glutathione, r, B.F. Polk, C.G. Hames tk of cancer. 980) Effect of ibetweer 2vivo. Cancer { 7,12-dimelhby selenium, t r 1 j J ) I Mutation Research, 154 (19S5) 49-67 Elsevier MTR 0719) 49 Current status of bioassays in genetic toxicology the dominant lethal assay A report of the U.S. Environmental Protection Agency Gene-Tox Program * Sidney Green \ Angela Auletta 2, Jill Fabricant '\ Robert Kapp 4, Madhu Manandhar 5, Ching-ju Sheu 6, Janet Springer 2 and Brad Whitfield 7 1 Bureau of Foods. U.S. Food and Drug Administration. H'ashtngton. DC 20201. - Health and Enriroomental Rerieie Dirision. U.S. Environmental Protection Agency. II'oshington, DC 20160. ' JSI Lakeside Lane, So. 100. Houston. TS 77SOS. 1 Btodynamtes. Inc.. E. Millstone, SJ OSS7\ ' Lederte Laboratories. American Cyanantid. Pearl Hirer, SY 10962. rt Food and Drug Administration, 11 o thington. PC 10 and ' Environmental Mutagen Information Center, Oak Ridge Sattonal Laboratory * *. Oak Ridge. TS J 7SS! (U.S. A, l (Received 14 December 1984) (Accepted IS December 19S4) Contents ! Summary.................................................. ............................................................................................................................................................. 50 I. Introduction.............................................................................................................................................................................................. 50 : A. History....................................... 50 B. Rationale.............................................................................................................................................................................................. 51 C. Types of studies................................................................................................................................................................................. 51 D. Selection of papers ......................... 51 II. Test description......................................................................................................................................................................................... 52 A. Cytogenetic basis of the dominant lethal effect............................................................................................................................ 52 B. Description of test animals........................................................................... 52 III. Interpretation of data.......................................................................................... 53 A. Presentation of data .......................................................................................................................................................................... 53 1. Dose-response cuiwes .................................................................................................................................................................. 53 2. Data transformations.................................................................................................................................................................. 53 3. Units of expression of data ........................................................................................................................................................ 53 B. Criteria for acceptability of data............... ............................................................................................................ ................... 54 C. Statistical evaluation.......................................................................................................................................................................... 54 D. Criteria for positive/negative conclusions.................................................................................................................................... 54 E Applicability of results tohaiard evaluation................................................................................................................................... 54 IV. Test performance....................................................................................................................................................................................... 61 Work Group Report prepared for the Gene-Tox Program (Office of Toxic Substances, Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC). The authors are members of the GeneTox Work Group on the Dominant Lethal Assay. Operated by Union Carbide Corporation under Contract No. W-7405-eng-26 with the U.S. Department of Energy, By acceptance of this article, (he publisher or recipient acknowledges the U.S. Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering this article. Although the review described in this article has been funded wholly or in part by the U.S. Environmental Protec tion Agency through interagency agreement DOE 40-112380. EPA No. 80-DX0953 to the Oak Ridge National Laboratory, it has not been subjected to the agency's re quired peer and policy review and, therefore, does not necessarily reflect the views of the agency and no official endorsement should be inferred. The protocols stated, sug gested use of the assay in a screening program, and research recommended should not be taken to represent agency policy on these matters. 'dnJ-l 110/85/S03.30 `lo 1985 Elsevier Science Publishers B.V. (lliomedtc.il Diwsion) iI 50 A. Number of chemicals tested................................................ B. Correlation with mammalian carcinogenicity data.......... V. Suggested protocol for testing.................................................. A. Treatment conditions .......................................................... B. Positive control and negative control................................. C. Dosage selection and number of doses.............................. D. Route of administration....................................................... E. Acceptable spontaneous background frequencies or rates F. Number of test animals....................................................... G. Proper collection of raw data............................................. VI. Conclusions . ............................................................................ References................................................................................................ 61 61 61 62 62 62 62 62 63 63 64 64 Summary The term dominant lethal may be defined as death of ihe heterozygote arising through multiple chromosomal breaks. The assay is generally conducted by treating male animals, usually mice or rats, acutely (1 dose), subacutely (5 doses), or over the entire period of spermatogenesis. Animals treated acutely or subacutely are mated at weekly intervals to females for a sufficient number of weeks to cover the period of spermatogenesis. Those treated for the entire spermatogenic cycle are mated for 1 or 2 successive weeks at the termination of treatment. Females usually are killed at 14 days of pregnancy and examined for the number of total implantations in the uterus, the number of implantations classified as early deaths, and, in some cases, the number of corpora lutea. The category of early death is the most significant index of dominant lethality. A total of 249 papers were reviewed and 140 chemicals were evaluated. Of the 140 chemicals, 65 were positive by the criteria used by the Work Group in evaluating each publication. The category of "positive" includes those responses of a borderline nature. 99 chemicals were declared negative. There is considerable overlap of chemicals in both categories, which accounts for the incongruity in the total number of chemicals tested and the number considered positive and negative. A total of 44 animal carcinogens have been tested in the dominant lethal assay, 26 of which were positive and 18 negative for a correlation of 59%. The role of the assay should be that of confirming positive results from lower tier chromosomal aberration-detecting systems (confirming in the sense of indicating the ability of the chemical to penetrate gonadal tissue and to produce cytogenetic damage). The dominant lethal assay should not be used as a risk assessment method. (I) Introduction It is now generally acknowledged that chemical agents pose hazards to the genetic machinery of the cell. Numerous methods of assessing gene, chromosomal, and primary DNA damage have been proposed. The dominant lethal assay is one of the few tests in which the mutagenic potential of an agent is examined directly in gametic tissue in an intact mammal. This is of importance in mutagenicity testing since there is little direct evi dence that genetic damage to somatic cells is re flective of similar damage in germ cells. The obvi ous advantage of utilizing in vivo mammalian data to extrapolate and assess safety in man need not be further amplified. (A) History A dominant lethal mutation is a genetic lesion that can occur in a gamete and, although not interfering with the gamete's ability to fertilize or be fertilized, is lethal to the resulting embryo (Bateman and Epstein, 1971). It has been shown that the genetic basis of these lethal events can be structural and/or numerical chromosomal anomalies (Brewen ct al.. 1975: Goldstein, 1977; tr 61 )r 61 | 62 i 62 62 ^ 62 62 ^ 63 b 63 64 t } 64 t | k ^ mliiple >r rats, icutely period weeks for the md. in dex of he 140 n. The i animal e for a er tier emical not be n data :d not lesion h not ize or nbryo hown an be omal 1977; Hitotsuniachi and Kikuchi, 1977; Kratochvilova, 1978; Matter and Jaeger, 1975). Brenneke (1937) first described dominant lethal mutations in mammals. In 1953,-Kaplan and Lyon used dominant lethal events "to assess genetic damage from radiation exposure, and Bateman (1966) proposed the use of dominant lethal mutations in screening for mutagens. (B) Rationale Dominant lethal assays may be particularly ger- mane to any discussion of human mutations since many recognizable human mutations are due to dominant autosomal traits (United Nations Scientific Committee, 1966). From evidence by Brewen et al. (1975), it was shown that the incidence of broken chromosomes at the first division corre lates with dominant lethality. It is expected that the broken chromosomes are eventually lost at anaphase, resulting in a monosomic embryo that subsequently dies in ulero. The dominant lethal assay theoretically can be used to screen agents that cause nondisjunction as well as those causing chromosomal breakage: in practice, the assay has been used only as a screen for chromosomal breakage in gametic cells. Domi nant lethal mutations are self-limiting by defini tion. because they are usually eliminated in the first generation and are not inherited. By them selves. these mutations are believed not to contrib ute to the genetic load. However, many mutagens produce both lethal and nonlethal chromosomal breakage. Reciprocal translocations and inversions are examples of the latter type breakage. Hence, the production of dominant lethal mutations could indicate the occurrence of other mutagenic events. (Cj Types of studies There are two fundamental approaches to per forming the dominant lethal test. In the first ap proach, adult male animals are dosed acutely (a single dose or a few doses within a few days) with sublethal concentrations of the agent. These treated animals are then mated sequentially (by weeks) with groups of untreated females. The results of the weekly matings can then indicate the specific stages of gametogenesis that are damaged and responsible for the resultant embryonic mortality. For example, matings of male mice during week 1 51 reveal effects on spermatozoa; during weeks 2 and 3, on spermatids; weeks 4 and 5, on spermato cytes; week 6, on differentiating spermatogonia; and after week 7. on stem cells (Bateman and Epstein, 1971). The timings are slightly diffe'rent in male rats, as weeks 1 and 2 represent effects on mature spermatozoa; weeks 3, 4, and 5, on spermatids; weeks 6, 7, and 8, on spermatocytes; weeks 9 and 10, on differentiating spermatogonia, and after week 11, on stem cells (Jackson, 1966). In these assays,'the females are sacrificed, usually between days 13 and 19 of gestation, and the uterine contents examined for evidence of dead implantations. In the second approach the male animals are dosed over the entire span of spermtno- and spermiogenesis (8 weeks in the mouse and 10 weeks in the rat) (Green et al.. 1975a.b. 1977). These treated males are then mated with groups of untreated females for 2 weeks. As in the afore mentioned protocol, the females are sacrificed, usually between days 13 and 19 of gestation, and examined for evidence of dead implantations. (D) Selection of papers There were a total of 450 papers listed under "dominant lethal" by the Environmental Mutagen Information Center(EMIC). Initially, each of these articles was examined briefly to exclude from the analysis those articles (1) that presented data as an abstract or only in descriptive form, (2) that were written in a foreign language for which no English translation was available. (3) that involved physi cal agents (i.e,, X-irradiation) and not chemical agents. Of the 450 papers, 201 were rejected based on the above criteria. Subsequently, the remaining 249 papers were submitted to the dominant lethal Work Group for evaluation. These articles were divided among the Work Group members. Each paper was evaluated by the individual assigned to that paper, and the results of that evaluation were presented to the entire Work Group for consensus of interpretation and final approval. The deliber ations of this final review resulted in an additional 104 papers being eliminated, utilizing the follow ing criteria. (1) The article did not involve a properly de fined chemical agent (mixtures). 30 i cn 52 (2) The results in the article were uninterpretable because of insufficient data or poor study design. (3) The article was a review and presented no new data. (4) The article 'was a symposium presentation without new data. (5) The rationale for dosage selection was not presented nor could the panel through available references determine the adequacy of the dosage, (6) The statistical evaluation utilized was inap propriate as determined by the Work Group statis tician. (7) The objectives of the article were outside the scope of the dominant lethal assay Work Group (e.g,, investigating the mechanism of dominant lethality) to the exclusion of what were considered adequate data for evaluation of the effects of a chemical. (8) The article could not be evaluated by the criteria adopted by the dominant lethal Work Group. The remaining 145 articles, which were found acceptable and met (he criteria, provide the data base (140 agents tested) for this report. (II) Test description IA) Cytogenetic basis of the dominant lethal effect Dominant lethality is measured by the frequency of embryonic deaths and is believed to arise from chromosomal abnormalities induced in the germ cells of the treated animals. In studies using known mutagens such as MMS, EMS, and TEM, the chromosomes of the resulting embryos have heen analyzed, and a good correlation between chro mosomal aberrations in early cleavage divisions and dominant lethality has been demonstrated (Brcwen ct al., 1975; Goldstein, 1977; Hitotsumachi and Kikuchi, 1977; Kraiochvilova, 1978; Matter and Jaeger, 1975). The embryonic deaths generally are found to occur either before or soon after implantation. Death before implantation, a preimplantation loss, is indicated by a reduction in the total number of implantations, and the death at implantation or soon thereafter, a postimplantation loss, is indi cated by an increase in the number of dead im plantations when compared with controls. The sig R&S 002400 nificance of the reduced count in total implanta tions is to be taken with reservation, because it does not provide a distinction between loss of fertilized eggs before implantation and loss of unfertilized eggs. The former -reflects gross chro mosomal aberrations that result in embryonic death before implantation. The latter reflects a toxic effect of treatment that reduces the germ cell's ability to fertilize or be fertilized. The in creased number of dead implantations is generally accepted as a good indication of dominant lethal ity. In practice, two types of dead implantations base been noted, early and late. The early dead implantation is referred to as a deciduoma or mole and is the result of an outgrowth of the uterus at the site of the implanting blastocyst that has failed to develop after implantation. The late dead im plantation is a dead embryo that has developed to a relatively advanced stage before death. In studies with known mutagens, it is observed that there is an increase of the early embryonic deaths with the do>age of treatment, whereas, the number of late embryonic deaths remains relatively stable irre spective of the treatment (Bateman and Epstein, 1971). Therefore, it is most likely that the early, but not the late, embryonic deaths are true repre sentatives of dominant lethality induced by chem icals. The classification of early versus late implan tations may vary from laboratory to laboratory, and. depending on the investigators, the postimplantation loss may represent only the early or both early and late embryonic deaths. (U) Description of rest animals A variety of organisms such as Drosophila, mouse, rat, hamster, and guinea pig has been used in the dominant lethal test. However, the mouse appears to be the most widely used, with the rat second (other organisms are rarely used). Various strains of outbred, inbred, or hybrid origin mice have been employed; the outbred strains appear to be used most widely. The test animals usually are selected on the basis of availability rather than sensitivity, and studies to determine the relative sensitivity of various strains are still very limited (Generoso, 1969; Generoso et ah, 1979). Females as well as males may be used as test animals, although the majority of tests are con ducted in males. Females appear less suitable as i. -..i an ise it I ;s of s of ;hro.'onic cts a germ e in rally t thal'.ions dead , nole is at died imd to dies 'e is the late rreein. riy, pre em !an- fy. im- or iila, sed use rat JUS lice ` to are tan ive ted esl tnas test animals in a system where successful fertiliza tion of the eggs is essential and where embryonic death is evaluated. A chemical administered to the female may interfere with the normal hormonal status of the animal and reduce the reproductive capacity as well as induce embryonic death due to nongenetic causes. A male animal can be analyzed by repeated matings with different females over a period of time, whereas a female can be studied for only one pregnancy. (Ill) Interpretation of data (A)Presentation of data (1) Dose-response curves A dose-response relationship was a primary criterion used in the evaluation of test data such as pregnancy rate, incidence of dead implantations, or total implantations. Statistically significant changes in these parameters, but without a dose-response relationship, were considered bio logically not significant unless other explanations could be offered (e.g., only one treatment group, an inadequate number of animals due to toxicity, or some other factor that clearly influenced the response). In interpreting data from the dominant lethal assay, the demonstration of a dose response should be one criterion in concluding that a chemical produced dominant lethality. Inasmuch as the dose response is influenced by a number of factors, some of which may not be considered by the genetic toxicologist prior to conducting an investi gation (e.g,, slope of dose-effect curve), one should exercise scientific judgment when such a response is not evident (e.g., a consistent one-dose effect over several weeks may well be significant). Ep stein et al. (1972) illustrate the concept of dose reversal for a number of chemicals, which is not an uncommon occurrence. In instances when a doserelated response is not evident but there is strong suspicion of activity, the study should be repeated. (2) Data transformation Data on the dominant lethal effect can be evaluated using various statistical tests. In one statistical approach, the data may be transformed before analysis. If preimplantation loss is de termined by counting corpora lutea, then the pre implantation loss of each female should be trans formed to the Freeman-Tukey arc sine before analysis by the Student's t test (Mosleller and Youtz, 1961). The index, dead implantations per pregnant female, should be transformed to the Freeman-Tukey square root before analysis by the t test (Generoso, 1969). The proportion, dead im plantations per total implantations, should be transformed to the,Freeman-Tukey arc sine be fore analysis by the t test. (3) Units of expression of data Some papers on the dominant lethal test classi fied dead implantations into early and late posiimplantation deaths, while other papers combined these into one category -- dead implantations. The Work Group recognizes that early postim plantation deaths are most likely to represent deaths due to genetic alterations, while late deaths are considered a negligible contribution and due primarily to nongenetic factors such as (he health of the dam. Therefore, in evaluating the dominant lethal papers, the Work Group decided to combine the categories of early and late deaths, when given separately, so that these classifications will be the same as those for dead implantations. In this wav, a degree of uniformity was maintained throughout the evaluation procedure without contributing any significant error. The main parameter used in evaluation of test data was the average number of dead implanta tions per pregnant female. However, additional information, such as percentage of animals preg nant and average number of total live implanta tions, had a bearing on whether the data associ ated with the parameter were accepted. In the evaluation of the data, the pregnant female was taken as the basic unit of experimentation. How ever, when data were not presented in this form, the given data were converted whenever possible to per pregnant female. The following units of expression were found acceptable by the Work Group and recommended for the dominant lethal assay. (1) Number of pregnant females/number of females mated; (2) number of fertile males/number of males mated; \1 CO N> 54 (3) average number of corpora lutea per preg nant female (when determined); (4) average number of implantations per preg nant female; (5) average number of preimplantation losses per pregnant femalet^hen determined); (6) average number of dead implantations (early and late) per pregnant female; (7) proportion of females with one or more dead implantations; (8) proportion of females with two or more dead implantations; (9) dead implantations/total implantations. The Work Group recognizes that in some in stances all of the above would be burdensome and recommends reporting data on the following as a minimum. (1) Number of pregnant females/number of females mated; (2) number of fertile males/number of males mated; (3) average number of total implantations per pregnant female; (4) average number of dead implantations (early and late) per pregnant female; (5) dead implanialions/ioial implantations. (B) Criteria [or acceptability of data The Work Group determined that as a mini mum the following be considered as criteria for accepting data for the dominant lethal assay; (1) A rationale should be presented that ad dresses the selection of the dosage; the maximum tolerated dose (MTD) is defined as the highest nonlethal dose that shows some degree of toxicity. (2) At least 2, but preferably 3 dosages should be used. (3) For proper documentation of negative data, results from a concurrent or recently tested posi tive control should be presented. (4) When using the acute or subacute dosage regimen, all stages of spermatogenesis should be sampled on a weekly basis to document negative results. (5) A sample size (number of pregnant females) sufficient to delect a doubling of the background frequency with a 95% probability is required to conclude that a result is negative. (6) Data should be evaluated on a "per preg nant female" basis and analyzed by a parametric statistical procedure on transformed data or a f comparable nonparametric procedure. (C) Statistical evaluation The Work Group has accepted dominant lethal papers in which any of the following procedures were employed in statistical evaluations: (a) Stu dent's / test, (b) analysis of variance, (c) chi-square test. It was the opinion of the Work Group that of these 3 tests, the chi-square test was applicable only for the fertility index. Comparable nonpara metric methods to the former two would be accep table. (D) Criteria for positive/negative conclusions In addition to criteria given in Section 111B. the following should be established to conclude that a result is negative; the number of pregnant females generated should allow detection of a doubling of the spontaneous frequency for a given species and/or strain, with a 95% probability. A 5% or less level of significance should be the criterion for concluding that a result is positive. (E) Applicability of results to hazard evaluation The dominant lethal test offers investigators an opportunity to assess mutagenic effects of chem ical agents. It is one of the few tests that evaluates the effect of test compounds on germinal tissues, is practical for routine testing procedures, and pro vides some information on the probability of a chemical affecting a future generation. The lest is suited to determine the potential transmissibility of mutagenic effects. In addition, when the domi nant lethal lest is conducted in the traditional way by mating on a weekly basis over the entire period of spermatogenesis, the stage(s) of spermatogenesis that may be most sensitive to a test compound can be determined as well. The role of the assay should be that of confirming positive results from lower tier chromosomal aberration-detecting systems (confirming in the sense of indicating the ability of the chemical to penetrate gonadal tissue and to produce chromosomal damage). A negative result in any of the mutagenicity tests is not necessarily final proof of the nonmutagenic nature of a com pound; the dominant lethal lest is no exception. R&S 002402 l ia trie w lata or a it iant lethal irocedures ^ s: (a) Stu- fc chi-square E up that of i applicable nonparabe accep- |- ions i 111B, the ude that a nt females oubling of :n species A 5% or ilerion for i # igators an of chem: evaluates i tissues, is . and prolility of a The test is missibility the doniitional way lire period iatogenesis pound can say should rom lower g systems : ability of t : i ue and to ttive result necessarily of a com.ception. | ^ f table 1 CHEMICAL TESTED IN THE DOMINANT LETHAL ASSAY Chemical (CAS No.) AF-2 (3688-53-7) Amethopterin (methotrexate) (59-05-2) Aminochlorohydrin (d) HCI (34839-14-0) Aminochlorohydrin (dl) HCI (34839-12-8) Aminochlorohydrin (l) HCI (34839-13-9) 8-Aminolevulinic acid HCI (5451-09-2) 1-Amino-2-naphthol-3. 6-disulfonic acid - Na salt (42579-07-7) 2-Amino-4-nitrophenol (99-57-0) 2-Amino-5-nitrophenol (121-88-0) 4-Amino-2-nitrophenol (119-34-6) Anovlar (8015-12-1) Aroelor 1242 (53469-21-9) Arivlor 1254 (11097-69-1) A/.athioprine (446-86-6) Bcnzonal (744-80-9) Butvlnilrosourea (869-01-2) Cadmium chloride (10108-64-2) Caffeine (58-08-2) Mutagen icity J,h -M M -R -R -R -M -R -R -R -R -M -R -R +M +M -M -M -M Carcino genicity c NR NR NR NR NR NR NR +L NR NR NR +L NR + + Capian (133-06-2) Chloramphenicol (56-75-7) Chlordane, AG (57-74-9) Chlordane, technical (12789-03-6) 1 -(2-ChloroethyI)-3-cyclohexyl1-nitrosourea (CCNU) (13010-47-4) C hlorohydrin, DL-a (96-24-2) + R.M -R +M -M +M -M M + R(M.F) -R +L * -t- L +L + NR 55 References Soares and Sheridan. 1975 Propping et al., 1972 Jones and Jackson. 1976 Jones and Jackson. 1976 Jones and Jackson. 1976 Arnold et al.. 1975 Palmer et al. 1979 _ Burnett et al.. 1977 Burnett et al.. 1977 Burnett et al., 1977 Badr and Badr. 1974 Green et al.. 1975a Green et al.. 1975a Clark. 1975 Eonshtein el al.. 1976 Propping et al., 1972 Gilltavod and Leonard, 1975 Adler. 1969. Epstein. 1970, Epstein et al., 1970a; Aeschbacher et al.. 1978 Collins; 1972a Sram. 1972 Arnold et al., 1977 Arnold et al.. 1977 Thompson et al., 1975 Jones and Jackson. 1976 33 </) o oro t* o co i , iff if 56 TABLE 1 {continued) Chemical (CAS No.) Cyclamaie sodium (139-05-9) Cyclohexylamine (10S-91-8) Cyclohexylamine sulfate (27817-50-1) Cyclophosphamide (50-18-0) DDT, technical p.p'-DDT (50-29-3) 2,4-DiaminoanisoIe (615-05-4) 2.4-Dtaminoantsole sulfate (39156-41-7) 2.5-Diaminoanisole (5307-02-8) 2.5-Diaminoarmole sulfate (42909-29-5) Diehlorvos (62-73-7) Dteldrin (60-57-1) Diethyladipaic (141-28-6) Di-2-ethyl-hexvhdipaie (DEHA) (103-23-1) Di-2-eihylhcxylphthalate (117-81-7) Dielhylhydroxylamine (3710-84-7) Diethylnitrosamine (DEN) (55-18-5) Diethyl sulfate (64-67-5) Difolatan (Captafol) (2425-06.)) Dihydroergotoxin methancsulfonate (8067-24-1) Dimethoxyethyl phthalatc (117-82-8) 1,1-Dimethylhydrazinc (57-14-7) Dimethylmercury (593-74-S) Dimelhylmyleran ( + / --) (33447-91-5) Dimelhylmyleran (mao) (33447-90-4) Dinilro-o-cresol J534.52-M Mutagen icity * b -M(F) -M +M -M M(F) -M(F) +M M M(F) M M M -R -R -R -R -R M " M(F) -M +M +M +M R -M -M R -f M M -M M +M +M -M Carcino genicity ' NR NR + + NR -rNR NR " NR NR NR + NR + + NR NR NR + NR NR NR NR References Machemer and Lorke. 1975 Cattanach and Pollard. 1971; Petersen et a!,, 1972 Lorke and Machemer. 1974; Machemer and Lorke, 1975 Rdhrborn, 1970; Rohrborn. 1970; Fritz et al.. 1973; Machemer and Lorke, 1975; Sram. 1976; Zhurko\ and Sram. 1978 Clark. 1974. Palmer et al.. 197T Burnett et al.. 1977 Sheu and Green, 1979 Burnett et a!,. 1977 Sheu and Green, 1979 Dean and 7`horpe. 1970: Dean and Blair. 1976 Dean et ah. 1975 Singh et al.. 1975 Singh et al.. 1975 Singh et al.. 1974 Legator et al.. 1978 Propping et al,, 1972 Malashenko. 1971; Malashenko and Surkova, 1973 Collins, 1972b Roberts and Rand. 1978 Singh et al.. 1974 Brusick and Matheson, 1976a Varma et al., 1974a Hemsworlh and Wardhaugh, 1977 Hemsworth and Wardhaugh. 1977 Nehci et al.. 1978 R&s 002404 f* B T V fe N Vt t t * table 1 (continued) Chemical (CAS No.)________ ___________ Uiqu.it (2764-72-9) Dimelhylnitrosamine (62-75-9) Dimethyl sulfoxide (67-68-5) Docloxythepin succinate (60003-04-5) EMS (ethyl methanesulfonaie) (62-50-0) Ergotamine tartrate (379-79-3) Ethanol (64-17-5) Ethylene oxide (75-21-8) Ethylenelhiourea (96-45-7) FD&C Red No. 2 (Amaranth) (915-67-3) Fenitrothion (122-14-5) Fluorescent whitening agent No. 1 (6416-68-8) Fluorescent whitening agent No. 2 (16090-02-1) Fluorescent whitening agent No. 3 (13863-31-5) Fluorescent whitening agent No. 4 (27344-41-8) Folpel (133-07-3) Fosfestcrol letrasodium (4719-75-9) Fotrin (37132-72-2) HEMPA (hexamethylphosphoramide) (680-31-9) Heptachlor epoxide (1024-57-3) I lexachlorobenzene (118-74-1) Mutagenicity'1, -M M M -M +M M(F) M(F) M M M(F) M M M M +M +M -M(F) +R +M -M M M -M -R -M -M -M -M -R R +M +M +M -M -R Carcinogenicity ` NR + * NR + NR -L 4 + NR NR NR NR NR NR NR NR NR + NR NR 57 References Pasi et al.. 1974; Anderson ei al., 1976a Propping et al., 1972 Aravindakshan et al.. 1975 Sykora et al., 1979 Ehling et al.. 1968: Generoso and Russell. 1969; Generoso, 1969; Soares and Crenshaw. 1974; Generoso el al.. 1974; Suter and Generoso, 1976; Arnold et al.. 1976a; Soares. 1976; Sram. 1976; Favor and Crenshaw. 197S Roberts and Rand. 1978 Badrand Badr. 1975 Machemer and Lork.e. 1975 Embree et al. 1977 Sehupbaeh and Hummler. 1977: Teramoto et al.. 1977; Teramoto et al.. 197S. Shirasu et al.. 1977 Arnold et a!.. 1976b Bencs et al.. 1975 Muller et al., 1975 Muller et al.. 1975 Muller et al., 1975 Muller el al., 1975 Collins, 1972b Ehling. 1979 Revazova and Radchenko, 1976 Sram et al.. 1970 Arnold et al., 1977 Simon et al.. 1979 R&S 002405 5S TABLE 1 (continued) Chemical (CAS No.) Hycamhone ' (3105-97-3) Hycanthone methanesulfonate (23255-93-8) ICR-170 (146-59-8) Indenopyridine derivative (67110-84-3) Imipramine (50-49-7) lsoniaaid (54-85-3) Isopropyl methanesulfonate (926-06-7) Lead subacetate (1335-32-6) LSD (50-37-3) Lyndiol (8015-14-3) 6-Mcrcapiopurinc (50-44-2) Methylhydrazine (60-34-4) Methyl mercuric chloride (115-09-3) zV-Methyl-jV'-nilro-/V-nitrosoguamdine (MNNG) (70-25-7) Mcthylnitrosourea (684-93-5) Methysergide hydrogen malcate (129-49-7) METEPA (57-39-6) Mi rex (2385-85-5) Mitonien (302-70-5) Mitomycin C (50-07-7) MMS (methyl methanesulfonate) (66-27-3) Mutagenicity * b -M +R -M M(F) -M -M -M +M M(F) M M M(E) - GP(F) +M -M M(F) +M - M.R -M.R +M - M(F) -M +M +M -R +M +M -M +M M M M M(F) M M Carcino genicity c NR * +L NR NR +L NR +* NR NR +L +L NR f + NR + 4* + -l" I rE* References Ray ei al,, 1975 Green et al,, 1973 Ehling et al,, 1968; Generoso, 1969 Matter el al.. 1979 Sykora et al,, 1979 Rdhrborn et a!., 1972 Mouischcn, 1969a; Generoso et al.. 1971; Ehling et al,, 1972; Generoso et al.. 1979; Suter and Generoso, 1976; Caine and Lyon. 1979 Varnta et al.. 1974b Sram et al., 1974 Badr and Badr. 1974 Generoso et al.. 1975; Schcncking and Frohberg. 1975 Brusick and Matheson. I976h Khera. 1973 Ehling et al.. 1968; Generoso. 1969 Propping et al.. 1972 Roberts and Rand. 1978 Epstein el al.. 1970c Khera el al.. 1976 Ehling, 1974 Ehling. 1971a; Ehling, 1971b Ehling et al., 1968; Moutschen, 1969b; Partington and Bateman. 1964; Bclilcs et al.. 1973; Machemcr and Lorke, 1975; Arnold et al.. 1976a; Dean and Johnstone. 1977; f? te r l if* fc- 30 C/> o o N-fa>. O cn r 59 TABLE 1 (continued) Chemical (CAS No.) Mutagen icity *s Carcino genicity c References MMS (methyl methanesulfonate) (66-27-3) (continued) * Myleran (busulfan) ft (55-98-1) Natulan (procarbazine hydrochloride) k (366-70-1) Nifurpipone * (24632-47-1) L- Nitrofurantoin (67-20-9) 4-N'itro-o-phenylene diamine (99-56-9) 2*Nitro-/>-phenylene diamine (5307-14-2) /V-Niirosoethylene thiourea (3715-92-2) Norelhistcrone acetate (51-98-9) - M( F) -M + M(F) +M +M -M -M R -R -R R -M M( f*) * + NR 4 +L NR NR Ceneroso. 1969: Ehling. 1977 Generoso ct al., 1971 Ehling. 1974: Roberts et a!.. 1979 Setnikar et al.. 1976 Setnikar et al.. 1976 Sheu and Green. 1979: Burnett et al.. 1977 Sheu and Green. 1979: Burnett et al,, 1977 Teramoto et al.. 1978 Rohrborn and Hansnunn, 1974 Oxamniquine (21738-42-1) Oxyprothepin dccanoutc (41931-S6-6) -M NR - M NR Ray et al.. 1975 S\ korj et al.. 1979 Paraquat (46S5-14-7) Patulin 975 (149-29-1) ib Phenylbutazone (50-33-9) /n-Phenylenediamine (108-45-2) o-Phenylenediamine (95-54-5) -M -M -M M M(P) +R -R -R NR - 1. NR NR NR Pasi et al.. 1974; Anderson et al . 1976a Roddv et al.. 1978 Machenier and Hess. 1971: Charles and Leonard. 1978: Machenier and Hess. 1973a Sheu and Green. 1979; Burnett et al.. 1977 Burnett et al., 1977 p-Phenylenediamine (106-50-3) - R - L Burnett et al.. 1977 fl-Phenyl-a-naphthylamine (90-30-2) M NR Brusick. and Mathoon. 1976c Polydimelhylsiloxane DC 360 (63148-62-9) -M NR Kennedy et al., 1976 Praziquantel (55268-74-1) -M NR Machenier and Lorke, 1978 Propylene oxide (75-56-9) -M + L Bootman ct al.. 1979 tf-Propyl meihancsulfonate (1912-31-8) Prothiaden (113-53-1) - M(F) +M -M NR NR Generoso et al.. 1971: Ehling et al.. 1972 Sykora et al.. 1979 Puromycin (53-79-2) -M NR Sram. 1972 Ronidazole (7681-76-7) -M NR Hite et al.. 1976 lii TABLE l (continued) Chemical (CAS No.) Mutagen icity *b Rubratoxin B (21794-01-4) Rubratoxin B, hydrogenated (31924-91-1) Saccharin (81-07-2) Saccharin, sodium (12S-44-9) Sodium bisulfite (7631-90-5) Sodium nitrite (7632-00-0) Sorbitol (50-70-4) Styrene oxide (96-09-3) +M --M +M +M -M M(F) -M(M.F) -M -R -M Triethylenemelamine (TEM) (5I-IS-3) + M(F) M(F) M CP M M M M M M M R M M(F) M -GP(F) tepa (545-55-1) +M M M 1,2,3.4-Tetrabromobutane (1529-68-6) M -R 2,3,7,8-TetrachIorodibenzo-/;-dioxin (1746-01-6) -R 5-Thio-D-glucose (20408-97-3) -R Thioproperazine-2-dimethylsuIfamido-10- -M (3-l-methyl-piperazinyl-4) propylphenolhiazine (2347-80-0) Thio-TEPA (52-24-4) +M M M M M Carcino genicity ' NR NR 4- + NR NR NR +L +L NR 4* NR NR + References Evans and Harbison, 1977 Evans and Harbison. 1977 Tezabwala and Gothoskar. 1977 Rao and Qureshi. 1972; Machemerand Lorke, 1973b; Machemer and Lorke, 1975 Generoso et al,, 1978 Teramoio et a!_J978 Maxwell and Newell, 1974 Fabry el al,, 1978 Sutcr and Generoso, 1976; Cattanach. 1959; Matter and Generoso. 1974; Cox and Lyon, 1975; Generoso et al.. 1977; Hastings et a!,. 1976; Hitolsumachi and Kikuchi, 1977; Green ei al., 1977; Soares and Sheridan, 1977; Schreiner and Steelman. 1977; Staub and Matter. 1977; Bateman, 1960; Sheu el al., J978; Generoso et al., 1971; Matter and Jaeger. 1975; Caine and Lyon, 1979 Sram et al., 1970; Sram, 1972; Sram and Zudova. 1973; Epstein et al.. 1970c Simon et al,, 1978 Khera and Ruddick. 1973 Majumdar el al.. 1979 Revazova et al,, 1975 Malashenko and Surkova, 1974a; Malashenko and Surkova. 1974b; Malashenko and Surkova, 1975; Sram. 1976; Malashenko et al.. 1978 02408 61 TABLE 1 (continued) Chemical (CAS No.) Trenimon (68-76-8) Mutagen icity *b + M(M,F) M(F) M(F) Carcino genicity ** References Rdhrborn. 1970: Machemer and Hess. 1973a; Machemer and Lorke. 1975 Tribromelhanol -M(F) NR Kaufman. 1977 m (75-80-9) o, a. a-Trifluoro-2-methyl- -R NR Beall. 1974 4'-nitro-m-proprionololuidide (13311-84-7) Triflupromazine (146-54-3) Trimethylphosphale + M NR Petersen and Legator. 1973; -M Ray et al.. 1973 +M + Epstein. 1970b (512-56-1) o-Toluenediamine -R NR Burnett et al.. 1977 (95-70-5) Trypaflavine + M(F) NR Busier et a! . 1977 (65431-33-6) Vinyl chloride (75-01-4) Vinyiidene chloride (75-35-4) -M R -R + Anderson et al.. 1976b; Short et al.. 1977 + L Short et al.. 1977 ' + . borderline response: considered positive in the estimation of correlation between mutagenicity and carcinogenicity: +,-. positive response in one experiment and negative in the other experiment, considered positive in the estimation of correlation 77; between mutagenicity and carcinogenicity: 4. positive; --, negative. h M. mouse; R, rat; GP. guinea pig; (F), female treated: (M.F). male and female treated in separate experiments; no entry, males treated. ' Source: Carcinogen list as reviewed by the EPA Gene-Tox Carcinogenesis Work. Group + . positive; + L, positive, limited: - L. negative, limited; NR, not reviewed; *. inconclusive. (IV) Test performance (A) Number of chemicals tested The number of chemicals considered in the dominant lethal assay at the time of this analysis is 140 (Table 1), These chemicals represent a spec trum of classes. It was not possible, however, due to overlap of chemicals among the various classes, to provide substantive information on testing re sults on a chemical-class basis. (B) Correlation with mammalian carcinogenicity data Table 1 summarizes the mutagenicity data available for the dominant lethal assay. Also pre sented are the results for chemicals that have been tested for carcinogenicity. Inspection of the table shows that of the 44 chemicals declared as carcinogens based on data from animals, 26 pro duced positive ( + ) or borderline ( + ) responses in the dominant lethal assay, for a 59% correlation. Consequently, this test is not viewed as one that could serve as a prescreen for carcinogenicity. (V) Suggested protocol for testing A dominant lethal test consists of 3 basic steps: (1) exposure of animals to a test compound, (2) mating of the treated animals to untreated animals of the opposite sex immediately after treatment, and (3) sacrifice and examination of the pregnant females to obtain the number of total, live, and dead implants. The dominant lethality is then as sessed on the basis of the number of dead implan tations per pregnant female. Since the test is usu ally conducted by treating males, the following discussion concerns only the male as the test animal. 62 (A) Treatment conditions Protocols for the dominant lethal test can be grouped into 3 types. One involves an acute treat ment followed by a succession of weekly matings, such as 6-8 weeks in mice and 8-10 weeks in rats. This method allows potential damage induced in the germ cells at a specific stage of development to be expressed through weekly mating. A second protocol involves a prolonged treatment, such as 6-8 weeks in mice and 8-10 weeks in rats, to cover the entire spermatogenic cycle, followed by 1-2 weeks of mating. This method allows potential damage induced in the germ cells at various stages of development to be expressed during the first week of mating but does not show the most sensi tive stage. A third protocol involves a short-term treatment (5 daily administrations) followed by a succession of weekly matings. The duration of treatment in this method is too short to cover the entire spermatogenic cycle, yet is often too long to allow precise determination of stage specificity. The first protocol is the method of choice for detecting stage specificity, while the second is best for evaluating prolonged effects of a chemical. The third protocol is used less often than the others; however, there is some evidence that a chemical can produce dominant lethality only after sub acute but not after acute administration (Green et al,, 1973). (B) Positive control and negative control A positive control is often included in testing to ensure that the experimental conditions are opti mal for the detection of dominant lethality. This control is necessary especially when the testing is conducted in a new laboratory, or when a new protocol or new species (strain) of animal is em ployed. In routine testing, a positive control may not always be necessary, but a concurrent negative control is always required because the spontaneous frequency of embryonic deaths is known to vary from species to species, strain to strain, and even from season to season. (C) Dosage selection and number of doses In the dominant lethal lest, the dosage em ployed is often selected on the basis of some biological effects reported in the literature or on the usage level for humans, sometimes with no obvious justification. Therefore, a negative re sponse simply may indicate a safe or no-effect level of the test compound without addressing the question of potential mutagenicity. It is essential, therefore, that a compound be tested not only at low but also at the MTD level so that the potential mutagenicity of a compound may be explored to the limit of technical ability. The proper de termination of the MTD level is critical, especially in prolonged treatment, because a compound with cumulative toxicity may complicate dosage selec tion. Ideally, an MTD should induce neither death nor sterility in the treated animals. One way to determine the MTD level is to treat a small num ber of test animals (e.g., 5 per dose level) for a range of doses (e.g,, 4 or 5) for Fweek or longer. The number of doses depends on previous in I ! j formation as regards the toxicity of the agent. The highest nonlethal dose that may slightly affect body weight gain can be selected as the MTD. If a compound has no detectable toxicity, then a dose level of 5 g/kg body weight may be taken as the MTD. Preferably, a minimum of 3 doses, includ ing the MTD. should be used in routine testing. e; fr W Pf co iff as re] IF, Jec ser ear fen tan eva Thi dew Ah the (D) Route of administration Chemicals can be and have been administered by a variety of routes: inhalation, gavage, intraperitoneal injection, or intravenous injection. Topical application, however, has not been used as a means of administering chemicals in the domi nant lethal assay. 33 e CO o o N> --*i O (E) Acceptable spontaneous background frequencies or rates In determining the acceptable background fre quencies or rates, a random analysis of control frequencies in terms of average number of dead implantations per pregnant female has been con ducted. This analysis was performed with data from the mouse. Sample sizes of at least 8 females per group were used, with those less than 8 not considered. With respect to the mouse, the average control rate varied from a low of 0.40 to a high of 0,80. It should be pointed out that each strain in this instance was of the random bred type. Control data from approximately 7500 individual females showed 4 out of 100 controls with more than 1 early death and 4 out of 1000 with more than 3 nific detC' becc mult out shou lion, nega level with (G)l T1 lions data R&S 002411 it *e, a negative re- s safe or no-effect '.out addressing the i -ity. It is essential, r tested not only at ^ o that the potential tay be explored to |` The proper de critical, especially ^ ; a compound with * icate dosage selec- i duce neither death mals. One way to treat a small num- dose level) for a 1 week or longer. ; < on previous inj of the agent. The tay slightly affect J as the MTD. If a xicity, then a dose ty be taken as the >f 3 doses, includi r testing. been administered ition, gavage. inuvenous injection, s not been used as icals in lire domi- ground frequencies e background frenalysis of control number of dead ale has been conormed with data at least 8 females e less than 8 not nouse, the average f 0.40 to a high of hat each strain in ored type. Control ndividual females vith more than 1 witli^Dre than 3 early deaths (Epstein et al., 1972). Although data from individual females were not examined by the Work Group, the average control rate among the various strains did not exceed 1 dead implantation per female. It would seem appropriate then to consider any control frequency beyond an average of 2 dead implantations per female in the case of the mouse as somewhat suspect and possibly to require a repeat experiment. (F) Humber of test animats The number of animals (young adults) sub jected to treatment is usually small, ranging from several to a few dozen per dose level. Generally, each treated male is mated to several untreated females (mating pairs identified), and the implan tations observed in pregnant females are then evaluated for evidence of dominant lethality. Therefore, the number of pregnant females per dose level is the critical parameter in this test. Although this number is determined primarily by the number of males subjected to treatment, it is also determined by such factors as the mating ratio of treated males to untreated females, the reproductive capacity of both male and female strains, and the effect of the chemical on the reproductive capacity of the treated male. Ideally, the number of pregnant females generated should allow detection, with a 95% probability, of a dou bling of the spontaneous frequency for a given species or strain of animals. However, a sample size is often selected on the basis of practicality, rather than statistical consideration. When no sig nificant increase over control frequency has been delected, the number of animals per dose level becomes a critical issue, since it determines the multiple of the control frequency that can be ruled out statistically. As a general rule, a sample size should be selected with due statistical considera tion. If that approach is not practical, when a negative result is obtained, one should specify the level of induced frequency that can be detected with the sample size employed. (G) Proper collection of raw data The number of both live and dead implanta tions in each pregnant female constitutes the raw data in the dominant lethal test. Therefore, any 63 factors that affect implantations could influence the test results. Examples of these factors are as follows: (1) Health of the pregnant female. The pres ence of intercurrent infection may affect implanta tion; therefore, the implantation data should be collected from healthy mothers. (2) Time of sacrifice of the females. Daily check for vaginal plugs or the performance of vaginal lavage permits accurate timing of mating and sub sequent sacrifice. Vaginal plugs can easily be ob served in the mouse. In the rat, vaginal lavage is the recommended technique. If vaginal smears are made or vaginal plugs observed^ the day on which sperm or plugs are observed is taken as day 0 of pregnancy, and females are killed on day 14. In routine testing, the females are all sacrificed at a fixed time, approximately 2 weeks following the midweek of cocaging. Therefore, the stage of preg nancy and thus the age of the implantations vary considerably (11-IS days), making the distinction between certain types of implantations somewhat difficult. The difficulty usually arises when a dosage level causes significant postimplantation loss and the day of sacrifice is such that a number of females are in the early stages of pregnancy (i.e., days 11-12). The distinction between early death and live implantations is more obscure in the early pregnant condition. It may be necessary to eliminate females in the early stage of pregnancy to reduce the margin of error. In some cases, a distinction can be made by dissecting the implan tation site and observing the embryo. (J) The classification of implantations. The im plantations are classified as live or dead, and dead implantations are often further classified as early or late. Since the identification of early and late deaths may rely on subjective judgment, welltrained personnel are required to conduct uterine analysis. (4) Counting of corpora lutea. Inasmuch as postimplantation loss (i.e., early death) is the most significant index of dominant lethality, the assess ment of preimplantation loss is not a requirement. If determination of preimplantation loss is a de sirable objective, one of two methods can be used. The first method makes use of corpora lutea counts and determines the difference between the number of corpora lutea and the number of implantations. This technique requires trained and skilled person nel. The rat allows an easier estimation of corpora lutea due to their greater size. The second method is to compare the -number of implantations per female in the treated and control groups. Any significant difference may be attributed to preim plantation loss. This approach is used more often for the mouse than for the rat. It should be pointed out that occasionally a reduction in the number of eggs released occurs, and then there is a significant reduction in implantations. In such cases, one may mistakenly implicate the chemical, because the assumption is that comparable numbers of eggs were released in treated and control groups. For tunately, this confusion does not occur often and can be avoided by performing counts of corpora lutea. (VI) Conclusions The dominant lethal assay is a whole animal test system that allows a chemical to be tested in vivo. Furthermore, it is one of the few mutagen icity tests that actually assesses the effect of chem icals on germ cells. This assay has sometimes been regarded as relatively insensitive (i.e., lack of re sponse to certain chemical mutagens); however, the germ cells within the whole animal obviously present a target very different than targets in in vitro systems. Physiological and pharmacological factors must be considered in evaluating results from the dominant lethal assay. It is not enough to highlight response differences between the domi nant lethal assay and in vitro assays, but investi gators should concentrate on the rationale un derlying each response. The failure to obtain a consistent response on repeated testing, particu larly when the weekly mating procedure is used, could be viewed as a weakness in this test. This problem is a statistical one brought about by the need to evaluate a large number of individual units (weekly values) for 5 or 6 indexes. The probability of obtaining a positive point increases as the num ber of points increase. It is not surprising that a number of chemicals have appeared positive but upon repeated testing were found to be negative. Analyses of variance across weeks have been used to evaluate results for all weeks in one spermato- genic cycle, reducing the number of individual h statistical tests run. The role of the dominant g lethal assay in a mutagenicity testing program P should be that of confirming results obtained in j other tests that are used to detect chromosomal r aberrations. Again, "confirming" is used to denote ! the ability of the agent to produce chromosomal t aberrations in germ cells that contribute to the genome of the offspring. From the analysis of the f Work Group, it appears that the dominant lethal j assay should not be employed in a carcinogenicity f testing program but can be used to detect potential ^ heritable germ cell damage. Although not as val- j uable as the translocation assay, the dominant lethal assay could provide important information for a subsequent heritable translocation test. References t Adler, l.-D. (1969) Humangenetik. 7. 137--14S. Ac.schbucher, H.U., H. Milon and H.P, Wurzncr (I97S) Muta tion Roi.. 57. 19.1-200. Anderson, D.. D.B. McGregor and I.F.H, Purchase (1976a) , Mutation Res., 40, 349-158. | Anderson, D., M.C.E. Hodge and I.F.H, Purchase (1976b) Mutation Res.. 40, 159-170, AravindaLshun. M,, P.S. Chauhan, A S. Aiyar and K. Sundaram (1975) Proe. Symp. Mutagenicity, Carcinogenicity and Teratogenicity of Chemicals 1975. Baroda, India, pp. 45-62. Arnold. D.W., G.L. Kennedy Jr., MX, Keplinger and J.C. Calandra (1975) Food Cosmct. Toxicol.. 11. 61-68. Arnold, D.W,. G.L. Kennedy Jr., M.L. Keplinger and J.C. Calandra (1976a) Toxicol. Appl. Pharmacol.. JS, 79-S4. I Arnold. D.W., G.L. Kennedy Jr., M.L. Keplinger and J.C. i Calandra (1976b) Food Cosmet. Toxicol., 14. 161-165. Arnold. D.W., G.L. Kennedy Jr.. M.L. Keplinger, J.C. Calandra and C J. Calo (1977) J. Toxicol. Environ. Health, 2. 547-555. Badr. F.M.. and R.S. Badr (1974) Mutation Res.. 26. 529-514. Badr, F.M., and R.S. Badr (1975) Nature (London), 251, 134-116. Busier, A.. M. Brucklacher, F. Nobis and G. Rohrborn (1977) | Hum. Genet., 40, 87-92. Bateman. A.J. (1960) Genet. Res.. I, 381-392. Bateman, A.J. (1966) Nature (London), 210, 205-206. Bateman. A.J.. and Epstein, S.S. (1971) Dominant lethal muta tions in mammals, in: A. Hollaender (Ed.). Chemical Mutagens: Principles and Methods for Their Detection. Vol. 2, Plenum, New York, pp. 541-568. Beall, J.R. (1974) Toxicol. Appl. Pharmacol.. 28, 301-312. Belilcs. R.P.. N. Korn and B.W. Benson (1973) Res. Commun. Chent. Pathol. Pharmacol.. 5. 713-724. Benes, V,, R.J. Sram and R. Tuscany (1975) J. Hyg. Epidemiol. Microbiol. Immunol., 19, 163-175. Bootman, J.. D.C. Lodge and H.E. Whalley (1979) Mutation Res., 67. 101-112. R&S 002412 imber of individual E e of the dominant \ ity testing program ^ results obtained in detect chromosomal t tg" is used to denote w oduce chromosomal E it contribute to the i t the analysis of the p the dominant lethal in a carcinogenicity d to detect potential ' .Ithough not as val- ssay, the dominant ponant information islocation test. 137-148. * Wurzner (1978) MuU- l-F.H. Purchase (1976a) F. rchasc (1976b) . Aiyar and K. 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